title: A low-density culture method of cerebellar granule neurons with paracrine support applicable for the study of neuronal morphogenesis.
abstract: Cerebellar granule neuronal cultures have been used to study the molecular mechanisms underlying neuronal functions, including neuronal morphogenesis. However, a limitation of this system is the difficulty to analyze isolated neurons because these are required to be maintained at a high density. Therefore, in the present study, we aimed to develop a simple and cost-effective method for culturing low-density cerebellar granule neurons. Cerebellar granule cells at two different densities (low- and high-density) were co-cultivated in order for the low-density culture to be supported by the paracrine signals from the high-density culture. This method enabled morphology analysis of isolated cerebellar granule neurons without astrocytic feeder cultures or supplements such as B27. Using this method, we investigated the function of a polarity factor. Studies using hippocampal neurons suggested that glycogen synthase kinase-3 (GSK-3) is an essential regulator of neuronal polarity, and inhibition of GSK-3 results in the formation of multiple axons. Pharmacological inhibitors for GSK-3 (6-bromoindirubin-3'-oxime and lithium chloride) did not cause the formation of multiple axons of cerebellar granule neurons but significantly reduced their length. Consistent results were obtained by introducing kinase-dead form of GSK-3 beta (K85A). These results indicated that GSK-3 is not directly involved in the control of neuronal polarity in cerebellar granule neurons. Overall, this study provides a simple method for culturing low-density cerebellar granule neurons and insights in to the neuronal-type dependent function of GSK-3 in neuronal morphogenesis.
authors.affiliation: Department of Human and Artificial Intelligence Systems, Graduate School of Engineering, University of Fukui, 3-9-1 Bunkyo, Fukui 910-8507, Japan.
authors.forename: Kenta
authors.initials: K
authors.lastname: Kubota
authors.forename: Takeshi
authors.initials: T
authors.lastname: Seno
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Konishi
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D016764:Cell Polarity; D002470:Cell Survival; D002531:Cerebellum / Q000166:cytology* / Q000196:embryology / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D051379:Mice; D009024:Morphogenesis; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D019899:Paracrine Communication
chemical_list: D038362:Glycogen Synthase Kinase 3
keywords: 6-bromoindirubin-3′-oxime; Axon; BIO; BME; CGN; CS; Cerebellar granule neuron; DMSO; GFP; GSK-3; LiCl; Low-density culture; Morphogenesis; PBS; Polarity; basal medium Eagle; calf serum; cerebellar granule neurons; days in vitro; dimethyl sulfoxide; div; glycogen synthase kinase-3; green fluorescent protein; lithium chloride; phosphate-buffered saline